Spontaneous adipogenic differentiation potential of adipose‑derived stem cells decreased with increasing cell passages

Abstract

Primary adipose-derived stem cells (ADSCs) are a mixture of cell types including preadipocytes having the ability to spontaneously differentiate into adipocytes. The aim of the present study was to compare the spontaneous adipogenic differentiation potential of ADSCs at different passages to determine whether it decreased with continuous cell passages. Mouse ADSCs (mADSCs) were harvested and cells from passages 1 to 5 were used for experiments. The proliferation of mADSCs at different passages was tested using the cell counting kit‑8 assay. Reverse transcription‑quantitative polymerase chain reaction was used to determine relative mRNA expression levels of CCAAT/enhancer binding protein α (Cebpa and C/EBPα) and peroxisome proliferator‑activated receptor γ (Pparg and PPARγ), and western blot analysis was used to investigate C/EBPα and PPARγ protein expression. The cells were cultured using DMEM. The fixed cells were then stained using Oil Red O on days 14 and 28, and the obtained extracted dye was monitored for absorbance. The 510 nm absorbance from passages 1 to 5 was observed to be statistically different. The relative expression levels of Cebpa and Pparg for mADSCs from passage 1 were significantly higher when compared with those for mADSCs from passages 2 to 5 on days 3, 5 and 7. However, no difference was identified in the expression levels of proteins C/EBPα and PPARγ for different passages. Although the mRNA expression levels of Pparg from passages 4 to 5 were significantly higher when compared with those from passage 1, the results of Oil Red O absorbance, mRNA expression levels of Cebpa, and the protein expression levels of C/EBPα and PPARγ exhibited no difference between mADSCs from passages 1 to 5 when cultured with induced adipogenic differentiation medium. Therefore, it was concluded that the spontaneous adipogenic differentiation potential of ADSCs decreased with continuous cell passages.

Engineering a 3D Vascularized Adipose Tissue Construct Using a Decellularized Lung Matrix

“…After 4 weeks of culture, hASCs cultured in differentiation media accumulated intracellular lipid droplets with a mixture of multilocular and unilocular cells ( Figure 3 C), while the slices cultured in growth media showed minimal differentiation ( Figure 3 B). Not surprisingly, the growth media promoted a small amount of spontaneous lipid accumulation, which we attribute to the low passage number of the hASCs used [ 45 ] and the high confluence [ 46 , 47 ]. Metabolic activity was also assessed in the tissue slices at the end of week 1 and week 4 ( Figure 3 D,E).…”

New Insight into Natural Extracellular Matrix: Genipin Cross-Linked Adipose-Derived Stem Cell Extracellular Matrix Gel for Tissue Engineering

“…Then, the undifferentiated state of ADSCs in the cell sheet was confirmed by the positive expression of CD105 and CD44 surface markers, as well as intracellular vimentin. Some studies revealed that ADSCs could differentiate into adipogenic [ 50 ], osteogenic, and chondrogenic lineages [ 51 ] after adding ascorbic acid [ 52 ] and long-term culturing [ 53 , 54 ], which has the risk of unwanted tissue formation. Hence, the undifferentiated state of ADSCs in cell sheets was confirmed using oil red O, Alizarin red, and Alcian blue staining following 10 days of culture with ascorbic acid or induction medium.…”

Engineering 3D Vascularized Adipose Tissue Construct using a Decellularized Lung Matrix

“…After 4 weeks, hASCs cultured in differentiation media accumulated intracellular lipid droplets with a mixture of multilocular and unilocular cells (Figure 3 C), while the slices cultured in growth media showed minimal differentiation (Figure 3 B). Not surprisingly, the growth media promoted a small amount of spontaneous lipid accumulation, which we attribute to the low passage number of the hASCs used [47] and the high confluence [48, 49]. Metabolic activity was also assessed in the tissue slices at the end of week 1 and week 4 (Figure 3 D-E).…”